Humanized antibody and process for preparing same

ABSTRACT

A humanized antibody is produced by process comprising the steps of: (a) selecting a specificity determining residue (SDR) of the complementarity determining region (CDR) of murine monoclonal antibody heavy chain and light chain variable regions; and (b) grafting said SDR to at least one of the corresponding amino acid sequences in human antibody variable regions.

FIELD OF THE INVENTION

The present invention relates to a process for preparing a humanizedantibody by grafting SDRs (specificity determining residues) in CDRs(complementary determining residues) of murine monoclonal antibody tohuman antibody and the humanized antibody prepared according to saidprocess.

BACKGROUND OF THE INVENTION

For preventing infectious diseases such as hepatitis B, there hasgenerally been used a method of administering immunoglobulins formed inblood plasma against a target antigen. However, the method has theproblems that the immunoglobulins generally have low specificity and maycontain contaminants.

Murine monoclonal antibody derived from mouse has been reported to havehigh affinity to antigen and is suitable for mass-production. However,repeated injection of murine monoclonal antibody induces an immuneresponse because the murine antibody is regarded as a foreign antigen inhumans (Shawler D. L. et al., J. Immunol., 135, 1530-1535 (1985)).

Accordingly, numerous efforts have been made to generate “humanizedantibody” by: grafting the CDR (complementarity determining region) ofmurine monoclonal antibody variable region which directly binds toantigens, to a human antibody framwork (CDR-grafting method); andreplacing the amino acid residues of the human antibody framework region(FR) that influence the CDR conformation with the amino acid residues ofmurine monoclonal antibody. The humanized antibody thus obtainedmaintains the affinity and specificity of original murine monoclonalantibody, and minimizes HAMA(human anti-mouse antibody) response inhumans (Riechmann et al., Nature, 332, 323-327 (1988); Queen C. et al.,Proc. Natl. Acad. Sci. USA, 86, 10029-10033 (1989); Nakatani et al.,Protein Engineering, 7, 435-443 (1994)). However, this humanizedantibody still causes problems when injected repeatedly into humans(Stephens et al., Immunology, 85, 668-674 (1995); Sharkey et al., CancerResearch, 55, 5935s-5945s(1995)).

Approximately 300 millions of world population carry hepatitis B virus(“HBV”) which may cause chronic infection, leading to cirrhosis andhepatocellular carcinoma (Tiollais P. and Buendia M. A., Sci. Am., 264,48 (1991)). The HBV envelope consists of three proteins, major proteincontaining S antigen, middle protein containing S and pre-S2 antigens,and large protein containing S, pre-S2 and pre-S 1 antigens (Neurath A.R. and Kent S. B., Adv. Vir Res., 34, 65-142 (1988)). These surfaceantigens have been known to play important roles in the process offorming antibodies against HBV in hepatitis patient. The pre-S1 region,in particular, is found on infectious viral particles (Heermann et al.,J. Viral., 52, 396-402 (1984)) and plays a role in attachment to cellsurface infection (Neurath et al., Cell, 46, 429 (1986); Pontisso etal., Viral., 173, 533, (1989); Neurath et al., Vaccine, 7, 234 (1989)).Thus a monoclonal antibody against the pre-S1 would be effective againstviral infection.

The present inventors have previously reported a murine monoclonalantibody (KR127) against HBV pre-S1 (Korean Patent No. 246128), a murinemonoclonal antibody KR127 gene encoding same (Korean Patent No. 250832)and a humanized antibody (HZKP 1271) of KR127 prepared by CDR-graftingmethod (Korean Patent No. 246128).

The present inventors have further endeavored to develop a humanizedantibody having minimized adverse immune response (HAMA response) aswell as enhanced affinity to antigen, and found that HAMA response canbe reduced when the amino acid residues of CDR of mouse antibody arereplaced with those of human antibody.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide aprocess for preparing a humanized antibody which is expected to showlower HAMA response and has higher affinity than humanized antibody ofthe prior art.

It is another object of the present invention to provide a humanizedantibody prepared according to said process.

It is a further another object of the present invention to provide a DNAencoding the heavy chain or light chain of said antibody and a vector 35comprising said DNA.

It is a still further object of the present invention to provide amicroorganism transformed with said vector.

In accordance with one aspect of the present invention, there isprovided a process for preparing a humanized antibody comprising thesteps of (a) selecting a specificity determining residue (SDR) of thecomplementarity determining region (CDR) of murine monoclonal antibodyheavy chain and light chain variable regions; and (b) grafting the aminoacid residues of said SDR to at least one of the corresponding aminoacid sequences in human antibody variable regions.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and features of the present invention willbecome apparent from the following description of the invention taken inconjunction with the following accompanying drawings; which respectivelyshow:

FIG. 1: the procedure for constructing an expression vector of achimeric heavy chain;

FIGS. 2A, 2B and 2C: collectively teach the nucleotide and amino acidsequence of the humanized 20 heavy chain variable region;

FIG. 3: the procedure for constructing an expression vector of achimeric light chain;

FIGS. 4A and 4B: the nucleotide and amino acid sequence of the humanizedlight chain variable region;

FIG. 5: the affinity to antigen of a humanized antibody having a heavychain CDR mutant;

FIG. 6: the procedure for constructing an expression vector of thehumanized antibody; and

FIG. 7: is a plot showing the results of analysis for MHC classII-binding peptide sequences in heavy chain variable regions of HuKR127and light chain variable regions of HuKR127, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The humanized antibody of the present invention may be prepared bygrafting the amino acid residues of SDR of murine monoclonal antibody tothe corresponding amino acid sequences in human antibody variableregions.

SDRs of the murine monoclonal antibody used in the present invention maybe determined by independently replacing each amino acid residue of CDRof the murine monoclonal antibody with alanine, selecting transformantswhich have lower affinity (k_(D)) to antigen than the original murineantibody and determining the replaced CDR amino acid residues of saidtransformants as SDRs.

Further, in order to enhance the affinity to antigen, the CDR residuesof a mouse antibody that increase the affinity and the frameworkresidues that influence the conformation of CDR loops may also begrafted to the corresponding sites of human antibody.

For example, the present invention describes a process for preparing ahumanized antibody for hepatitis B virus (HBV) pre-S1 by using murinemonoclonal antibody KR127 (Korean Patent No. 250832) as follows:

After selecting SDR amino acid residues, which play important roles inbinding with antigen, from CDR of the murine monoclonal antibody KR127heavy and light chains, chimeric heavy chain and chimeric light chaingenes may be prepared by combining either the variable region of ICR127antibody heavy chain with the constant region (C₁ 1) of human antibodyor the variable region of KR127 antibody light chain with the constantregion (CO of human antibody.

SDRs of the murine monoclonal antibody for HBV pre-S 1 are determined byreplacing each amino acid residue of CDR HCDRI (aa 31-35), HCDR2 (aa50-65) and HCDR3 (aa 95-102) of the heavy chain (SEQ ID NO: 2) and CDRLCDR1 (aa 24-34), LCDR2(aa 50-56) and LCDR3(aa 89-97) of the light chain(SEQ ID NO: 4) of the murine monoclonal antibody KR127 with alanineaccording to the alanine scanning mutagenesis method and selecting theamino acid residues (SDRs) whose replacement with alanine bring aboutmore than 3 times reduction in the affinity to antigen(KD) as comparedwith the original murine antibody. Throughout this description, aminoacid residue number is assigned according to Kabat numbering scheme(Kabat, E. A. et al, Sequences of Proteins of Immunological Interest.National Institute of Health, Bethesda, Md., 1991).

Examples of preferred SDR include tryptophan at position 33 (it isrepresented as “Trp33”), Met34, and Asn35 of HCDR1; Arg50, Tyr52, andPro52a of HCDR2; Glu95, Tyr96, and Glu98 of HCDR3 of the murinemonoclonal antibody KR127 heavy chain; Leu27b, Tyr27d, Ser27e; Asn28,Lys30, Tyr32, and Asn34 of LCDR1; Leu50 and Asp55 of LCDR2; and Va189,Gln90, Gly91, Thr92, His93, Phe94, Pro95, and Gln96 of LCDR3 of themurine monoclonal antibody KR127 light chain.

The humanized antibody of the present invention can be prepared bygrafting one. or more SDRs determined as above onto the human antibodyheavy chain and/or light chain. The human antibody heavy chain which maybe used in the present invention is human heavy chain DP7-JH4 consistingof human immunoglobulin germline VH gene segment DP7 (Tomlinson et al.,J. Mol. Biol., 227, 776-798, 1992) and JH4 segment (Ravetch et al.,Cell, 27, 583-591, 1981). The human antibody light chain which may beused in the present invention is human light chain DPK12-JH4 consistingof human immunoglobulin germline VK gene segment DPK12 (Cox et al., Eur.J. Irnmunol., 24, 827-836 (1994)) and JH4 segment (Hieter et al., J.Biol. Chem., 257, 1516-1522 (1982)).

The humanized heavy chain of the present invention may be prepared bygrafting at least one of Trp33, Met34, and Asn35 of HCDR1; Arg50, Tyr52,and Pro52a of HCDR2; Glu95, Tyr96, and Glu98 of HCDR3 of the murinemonoclonal antibody KR127 heavy chain to the corresponding amino acidsequences in human antibody heavy chain. The inventive humanized lightchain may be prepared by grafting at least one of Leu27b, Tyr27d,Ser27e; Asn28, Lys30, Tyr32, and Asn34 of LCDR1; Leu50 and Asp55 ofLCDR2; and Va189, Gln90, Gly91, Thr92, His93, Phe94, Pro95, and Gln96 ofLCDR3 of the murine monoclonal antibody KR127 light chain to thecorresponding amino acid sequences in human antibody light chainDPH1241(4.

Moreover, the affinity to antigen of the humanized antibody can beenhanced by the follow substitutions:

(a) the amino acid residue at position 32 in HCDR1 of the modified humanheavy chain DP7-JH4 by Ala;

(b) the amino acid residue at position 97 in HCDR3 of the modified humanheavy chain DP7-JH4 by Arg or Ala;

(c) the amino acid residue at position 98 in HCDR3 of the modified humanheavy chain DP7-JH4 by Val; and

(d) the amino acid residue at position 102 in HCDR3 of the modifiedhuman heavy chain DP7-3H4 by Arg or Ala.

In addition, Ala71 and Lys73 in framework region 3 in the heavy chainvariable region of KR127, which affects the conformation of the CDRloop, may further be grafted to human heavy chain DP7-JH4. Also, Leu36and Arg46 in framework region 2 in the light chain variable region ofKR127, which affects conformation of CDR loop, may be further grafted tohuman light chain DPH12-3K4.

The heavy chain variable region of humanized antibody of the presentinvention has the amino acid sequence of SEQ ID NO: 2, preferablyencoded by the nucleotide sequence of SEQ ID NO: 1 and the inventivelight chain variable region of humanized antibody has the amino acidsequence of SEQ ID NO: 4, preferably encoded by the nucleotide sequenceof SEQ ID NO: 3.

The humanized antibody heavy chain and light chain of the presentinvention may be encoded by a gene comprising a nucleotide sequencededuced from the humanized antibody heavy chain and light chainaccording to the genetic code. It is known that several different codonsencoding a specific amino acid may exist due to the codon degeneracy,and, therefore, the present invention includes in its scope allnucleotide sequences deduced from the humanized antibody heavy chain andlight chain amino acid sequence. Preferably, the humanized antibodyheavy chain and light chain gene sequences include one or more preferredcodons of host cell.

The humanized antibody consisted of the humanized heavy chain HuKR127HCof the present invention and humanized light chain HZKR127I prepared byCDR-grafting has an affinity to antigen of about over 50 times higherthan that of the humanized antibody HZKR1271.

The humanized antibody consisting of the humanized heavy chain HuKR127KCof the present invention and humanized light chain HZKR127I prepared byCDR-grafting has an affinity to antigen equal to that of the humanizedantibody HZICR127I.

The genes of humanized antibody heavy chain and light chain thusprepared may be inserted to pdCMV-dhfrC-HAV6 vector (KCTC 10028BP) toobtain an expression vector pdCMV-dhfrC-HuKR127 which can express bothhumanized antibody heavy chain HuKR127HC and light chain HZKR127I. Theexpression vector of the present invention may be introduced intomicroorganism, e.g., E. coli DH5a according to a conventionaltransformation method to obtain transformants E. coliDH5a/pdCMV-dhfrC-HuKR127. The transformants E. coli DH5apdCMVdhfrC-HuKR127 was deposited on Mar. 13, 2002 with the KoreanCollection for Type Cultures(KCTC)(Address: Korea Research Institute ofBioscience and Biotechnology(KRIBB), #52, Oun-dong, Yusong-ku, Taejon,305-333, Republic of Korea) under the accession number, KCTC 10198BP, inaccordance with the terms of Budapest Treaty on the International . . .Recognition of the Deposit of Microorganism for the Purpose of PatentProcedure.

Meanwhile, CHO/HuKR127, CHO (Chinese hamster ovary) cell linetransfected with vector pdCMV-dhfrC-HuKR127, was deposited on Mar. 13,2002 with the Korean Collection for Type Cultures(KCTC) under theaccession number, KCTC 10199BP, in accordance with the terms of BudapestTreaty on the International Recognition of the Deposit of Microorganismfor the Purpose of Patent Procedure.

The humanized antibody HuKR127 of the present invention produced byculturing the CHO/HuKR127 cell line has a higher affinity to antigen andis expected to reduce HAMA (human anti-mouse antibody) response to agreater extent than the conventional antibody prepared according to theCDR-grafting method.

Accordingly, the humanized antibody of the present invention can be usedin preventing hepatitis B virus infection and treating chronic HepatitisB.

Thus, for preventing hepatitis B virus infection and treating chronicHepatitis B, a pharmaceutical formulation of the inventive humanizedantibody may be prepared in accordance with any of the conventionalprocedures.

The pharmaceutical composition of the present invention can beadministered via various routes including intravenous and intramuscularintroduction. It should be understood that the amount of the activeingredient actually administered ought to be determined in light ofvarious relevant factors including the condition to be treated, thechosen route of administration, the age, sex and body weight of theindividual patient, and the severity of the patient's symptom; and,therefore, the above dose should not be intended to limit the scope ofthe invention in any way.

The following Examples are intended to further illustrate the presentinvention without limiting its scope.

Example 1 Preparation of Mouse/Human Chimeric Heavy Chain Gene

The gene encoding leader sequence and the yl constant region of thehuman antibody heavy chain were separately prepared by carrying out PCRusing pCMV-HKR127HC (Korean Patent No. 246128, KCTC 0531BP) as atemplate and a primer set of Ryu94 (SEQ ID NO: 5) and HUR43-1 (SEQ IDNO: 6) or HUR46-1 (SEQ ID NO: 9) and HUR31 (SEQ ID NO: 10). The geneencoding heavy chain variable region of the murine monoclonal antibodyKR127 was prepared by carrying out PCR using pKR127H (Korean. Patent No.250832, KCTC 0333BP) as a template and primers HUR44-1 (SEQ ID NO: 7)and HUR45-1 (SEQ ID NO: 8).

Ryu94: 5′-GAG AAT TCA CAT TCA CGA TGT ACT TG-3′ HUR43-1:CTG CAG CTG GAC CTG ACT CTG GAC ACC ATT-3′ HUR44-1:5′-CAG GTC CAG CTG CAG CAG TCT GGA CCT GAA CTG-3′ HUR45-1:5′-TGG GCC CTT GGT GGA GGC TGC AGA GAC AGTGAC-3′ HUR46-1:5′-GCC TCC ACC AAG GGC CCA TCG GTC TTC CCC CTG-3′ HUR31:5′-CAG CGG CCG CTC ATT TAC CCG GGG ACA G-3′

Each PCR reaction was carried out using 10 ng of template, 1 ie of eachprimer (50 ppm), 0.5 p.t of _(Pfu) DNA polymerase (Promega), 4 a of 2.5mM dNTPmix and 5, ull of 10×Pfu reaction buffer solution. Afterpre-denaturation at 95 t for 5 minutes, a PCR cycle was repeated 25times, the cycle being composed of 95 t for 30 sec., 50° C. for 30 sec.and 72° C. for 45 sec. After annealing the DNA fragment obtained byusing primers Ryu94 and HUR43-1, the DNA fragment obtained by usingprimers HUR44-1 and HUR45-1, and the DNA fragment obtained by usingprimers HUR46-1 and HUR31 were recombined by conducting recombinant PCRusing primers Ryu94 and HUR31. The recombinant PCR reaction was carriedout using the same reaction buffer solution as used above. Afterpre-denaturation at 95 t for 5 minutes, a PCR cycle was repeated 30times, the cycle being composed of: 95 t for 30 sec., 50t for 30 sec.and 72 t for 60 sec., and finally, the extension reaction was carriedout at 72 t for 5 min.

The chimeric heavy chain gene thus prepared was cleaved with EcoRI(GAATTC) and Ndel (GCGGCCGC) and inserted at the EcoRTINdel section ofvector pcDdA (plasmid which is removed Apal site in the multiple cloningsite of pcDNA received from Invitrogen), to obtain, vectorpcDdAchKR127HC (FIG. 1). The base sequence of the chimeric heavy chainvariable region gene (KR127VH) was confirmed by DNA sequence analysis(FIG. 2).

Example 2 Preparation of Mouse/Human Chimeric Light Chain Gene

The gene encoding reader sequence and the constant region of the humanantibody light chain were each prepared by carrying out PCR usingpKC-dhfr-FIKR127 (Korean Patent No. 2000-33008, KCTC 0529BP) as atemplate and a primer set of Ryu86 (SEQ ID NO: 11) and HUR48 (SEQ ID NO:12) or HUR51 (SEQ ID NO: 15) and CK1D (SEQ ID NO: 16).

The gene encoding light chain variable region of the murine monoclonalantibody KR127 was prepared by carrying out PCR using pKR127K (KoreanPatent No. 250832, KCTC 0334BP) as a template and primers HUR49 (SEQ IDNO: 13) and HUR50 (SEQ ID NO: 14).

Ryu86: 5′-CAA AGC TTG GAA GCA AGA TGG ATT CA-3′ HUR48:5′-CAA GAT ATC CCC ACA GGT ACC AGA TAC-3′ HUR49:5′-TGT GGG GAT ATC TTG ATG ACC CAA ACT-3′ HUR50:5′-CAC AGA TCT TTT GAT TTC CAG CTT GGT-3′ HUR51:5′-ATC AAA AGA TCT GTG GCT GCA CCA TCT-3′ CK1D:5′-GCG CCG TCT AGA ATT AAC ACT CIC CCC TGT TGAAGC TCT TTG TGA CGG GCG AACTCAG-3′

Each PCR reaction was carried out according to the method described inExample 1 except that primers Ryu86 and CK1D were used to ligate theannealed DNA fragments obtained by PCR reactions.

The chimeric light chain gene thus prepared was cleaved with HindlII(AAGCTT) and Xbal (TCTAGA) and inserted at the HindMIXbal section ofvector pBluescript KS, to obtain a recombinant plasmid. Subsequently,the recombinant plasmind was cleaved with Hindilll and Apal and insertedat the HindlrII Apal section. of vector pCMV-dhfr (KCTC 8671P), toobtain plasmid pKC-dhfr-chKR127 (FIG. 3). The base sequence of thechimeric light chain varible region gene (KR127VK) was confirmed by DNAsequence analysis (FIG. 4).

Example 3 Mutation of CDR. of Chimeric KR127 Antibody Heavy Chain byAlanine Injection

To examine whether each amino acid residue of KR127 heavy chain HCDR1(aa 31-35), HCDR2(aa 50-65) and HCDR3 (aa 95-102) binds to antigen, PCRreaction was carried out using vector pcDdA-chKR127HC as a template toprepare a modified gene, wherein an amino acid residue of CDR wasreplaced with alanine (the replaced amino acid residue No. was indicatedas Kabat number) (see FIG. 2).

A forward primer YMOO1N of SEQ ID NO: 17 was designed to provide thesequence corresponding to the reader sequence at the 5′-end of thechimeric heavy chain gene and EcoRI restrition site, and a reverseprimer YM003 of SEQ ID NO: 18 was designed to have the sequencecorresponding to the N-terminal downstream of CH1 domain of human heavychain gene and Apal restriction site.

YMOO1N: 5′-CCG GAA TTC ACA TTC ACG ATG TAC TTG-3′ YM003:5′-TGC CCC CAG AGG TGC T-3′

The 5′-end primer ym257 of SEQ ID NO: 19 (corresponding to nucleotideNos. 80 to 112 of SEQ ID NO: 1) was designed to replace Ser31 of HCDR1with alanine (S31A) and the 3′-end primer YM258 of SEQ ID NO: 20(corresponding to nucleotide Nos. 101 to 71 of SEQ ID NO: 1), to replaceAGT (coding for Ser) of nucleotide Nos. 91 to 93 of HCDRI gene with GCT(coding for alanine).

Each PCR reaction was carried out according to the method described inExample 1 except that primer sets, YMOO1N and YM258; and ym258 andYM003, were used and also that primers YMOO1N and YM003 were used torecombine the annealed DNA fragments obtained by PCR.

The chimeric light chain gene thus prepared was cleaved with EcoRI andApal and inserted at the EcoRTIApal section of vector pcDdA-chICR127HCprepared in Example 1, to obtain peDdA-chICR127HC-S31A. The basesequence of the humanized antibody heavy chain variable region gene wasconfirmed by DNA sequence analysis. Vectors containing mutants thusprepared are shown in Table 1.

In Table 1, primer and mutation positions are numbered based on the basesequence of SEQ ID NO: 1.

TABLE 1  primer mutation CDR primer position position mutant vector Fym257 80-112 91-93 S er CACTI-?+0 R YM258 101-71 Ala(GCT)pcDdA-chICR127HC-S31A F yrn259 83-112 Sre CTCT1-?+0 R YM260 106-73 94-96Ala(GCT) pcDdA-chKR127HC-S32A F ym261 86-117 Trp(TGG)-pcDdA-chKR127HC-1Y33A HCDR1 R YM262 108-76 97-99 Ala(GCG) F ym263 90-118Met(ATCF)- R YM264 111-79 100-102 Al a(GCG) pcDdA-chKR127HC-M33A F ym26594-120 Asn(AAC)- R ym266 112-81 103-105 Ala(GCC) pcDdA-chKR127HC-N35A FYIV1221 139-174 Arg(CGG)-′ pcalA-chICR.127HC-R50A R YM222 158-128148-150 Ala(GCC) F YM225 143-178 151-153 I I e(A 1 1)-pcDdA-chKR127HC-I 51A R YM226 162-131 Ala(GCT) F YM227 145-180Tvr CT′AT)- R YM228 165-135 154-156 Ala(GCT) pcDdA-chKR127HC-Y52A Fym229 148_181 Pri-dn′T 1-?+0 R YM230 167-136 157-159 AI a(GCT)pcDdA-chICR127HC-P52aA F ym231 150-186 G lv(GGA)- R YM232 173-145160-162 A 1 a(GCA) pcDdA-chICR127HC-G53A HCDR2 F vm233 152-188Asn(C1AT4- R YM234 176-144 163-165 Ala(GCT) pcDdA-chKR127HC-D54A F ym235155-193 Glv(GGA)-?+0 R YM236 178-146 166-168 A Ia(GCA)peDdA-chKR127HC-G55A F ym2 3 7 158-194 A cn(GAT′-?+0 ym238 184-149169-171 A I a(GCT) pcDdA-chKR127HC-D56A F ym239 160-195 Thr(M. 1 ′1- Rym240 185-150 172-174 A 1 a(GCT) pcDdA-chICR127HC-T57A F yrn241 164-196Acn(A AC1- R ym242 187-150 175-177 Ala(GCC) pcDdA-ch1CR127HC-N58A FYM207 286-317 Glu(GAG1- R YM208 305-274 295-297 A 1 a(GCG)pcDdA-chICR127HC-E95A F YN1209 289-316 Tvr(TAC′.)-. R YNI210 307-276298-300 A 1 a(GCC) pcDdA-chKR127HC-Y96A F YM211 292-318 Asn(GAC)-?+0HCDR1 R YN1217 111-279 Al a CGCG) F YM213 296-321 Gln(CIAG)-. R YM214315-285 304-306 A I a(GCG) pcDdA-chKR127HC-E98A F YM255 303-327Tvr(TAO-. R YM256 319-289 310-312 Ala(GCC) peDdA-chKR127HC-Y102A

Test Example 1 Expression of Chimeric Antibody Having a Modified HeavyChain and its Affinity to Antigen (Step 1) Expression of ChimericAntibody

COS7 cells (ATCC CRL-1651) were seeded to DMEM media (GIBCO) containing10% bovine serum and subcultured in an incubator at 37° C. under anatmosphere of 5% CO₂. 1×10⁶ cells thus obtained were seeded to the samemedia and incubated at 37° C. overnight. Thus, 5 lig of plasmidpICC-dhfr-chKR.127 (expressing chimeric light chain) obtained in Example2, 5 jig of plasmid obtained in Example 3 were diluted withOPTI-1VLEMAGLBCO) to 800 ge. 50 of Lipofectamine (GIBCO) were dilutedwith the same solution to 800 _(I)a. The resulting solutions were addedto a 15 rae tube, mixed and then, kept at room temperature for more than15 minutes. Meanwhile, COS7 cells incubated as above were washed threetimes with OPTI-MEM I. Then, 6.4 me of OPTI-MEM I was added to theDNA-Lipofectamine mixture and the resulting solution was evenlydistributed on the COS7 cells, which were cultured for 48 hours in a 5%CO₂ incubator to obtain a supernatant. The resulting solution wassubjected to sandwich ELISA analysis using anti-human IgG (Sigma) as acapture antibody and anti-human antigen (Fc-specific)-horseradishperoxidase (PIERCE) as a secondary antibody to confirm the expression ofthe chimeric antibody.

(step 2) Affinity to Antigen

150 ng of HBV recombinant antigen GST-pre-S1(1-56) (H. S. Kim and H. 3.Hong, Biotechnology Letters, 17, 871-876 (1995)) was coated to each wellof a microplate and 5 ng of the supernatant obtained in Step 1 was addedto each well. The resulting solution was subjected to indirect ELISAusing the same secondary antibody as used in step 1, followed bymeasuring the absorbance at 450 nm. Further, the affinity to antigen(K_(D)) of each modified heavy chain was determined by competitive ELISAmethod (Ryu et al., J. Med. Viral., 52, 226 (1997)) and compared withthat of pCK-dhfr-chKR.127 containing wildtype chimeric heavy chain. Theresult is shown in Table 2.

TABLE 2 KD CDR Mutant (nM) WT  11.0 ± 1.664 ii1 S31A 14.67 ± 2.386 S32A8.455 ± 0.840 W33A >10000 M34A >10000 N35A >10000 H2 R50A    >10000 •I51A 12.8 ± 1.05 Y52A 276.8 ± 23.60 P52aA 170.3 ± 5.318 G53A 7.697 ±0.980 D54A 1.663 ± 0.477 G55A 5.766 ± 0.211 D56A 6.59 ± 1.09 T57A 13.68± 4.016 N58A 1.568 ± 0.085 H3 E95A • >10000    Y96A >10000 D97A 0.57 ±0.03 E98A 64.2 ± 7.78 Y102A 3.581 ± 0.457

As shown in Table 2, the affinities to antigen of the mutants obtainedby replacing Trp33, Met34, or Asn35 of HCDR1; Arg50, Tyr52, or Pro52a ofHCDR2; Glu95, Tyr96, or Glu98 of HCDR3 with alanine were more than 3times lower than that of wild type. However, a mutant having alaninesubstituting for Asp97 or Tyr102 residue of HCDR3 exhibited an enhancedaffinity to antigen.

Example 4 Preparation of HCDR3 Mutants and Their Affinities to Antigen

(step 1) D97R and E98V Mutants

Each mutant was prepared by replacing Asp97 or Glu98 of HCDR3 witharginine as a positively charged amino acid (it is represented as“D97R”) or valine as a neutral amino acid (it is represented as “E98V”)according to the site-directed mutagenesis as used in Example 3. Vectorscontaining mutants prepared as above are shown in Table 3.

TABLE 3 mutation CDR primer position position mutant vector HCDR3 R P1312-279 301-303 Asp(GAC)→ pcDdA-chKR127HC-D97R F P2 295-326 Arg(CGG) RPC 312-279 301-303 Asp(GAC)→ pcDdA-chKR127HC-D97V F P4 295-326 Val(GTT)R P5 312-279 304-306 Glu(GAG)→ pcDdA-chKR127HC-E98R F P6 295-326 Arg(CGGR P7 312-297 304-306 Glu(GAG)→ pcDdA-chKR127hc-E98V F P8 295-326Val(GTT)

Then, each mutant thus obtained was measured for its affinity to antigenin according to the method described in Test Example 1 and compared withthat of the wild type.

As shown in FIG. 5, the affinity to antigen of D97R was more than 3times higher than that of the wild type, which the affinity to antigenof E98V, more than 4 times higher than that of the wild type. However,mutant E98R showed a low affinity to antigen.

(Step 2) D97R/E98V Mutant

To prepare D97R/E98V mutant containing both D97R and E98V, which werefound to be mutants having high affinity to antigen, PCR reaction wascarried out using pcDdA-chICR127HC-D97R which contains D97R gene as atemplate and primers P7 and P8.

Then, the D97R/E98V mutant thus obtained was measured for its affinityto antigen in according to the method described in Test Example 1.

As shown in FIG. 5, the affinity to antigen of D97R/E98V was more than15 times higher than that of the wild type.

(Step 3) D97R/E98V/Y102A Mutant

To prepare D97R/E98V/Y102A mutant containing D97R, E98V and Y102A, PCRreaction was carried out using pcDdA-chICR127HC-RV containing D97R/E98Vas a template and primers YM255 and YM256.

Then, the D97R/E98V/Y102A mutant (hereinafter “RVAA”) thus obtained wasmeasured for its affinity to antigen in according to the methoddescribed in Test Example 1.

As shown in FIG. 5, the affinity to antigen of D97R/E98V/Y102A wassimilar to that of D97R/E98V.

(Step 4) D97R/E98V/Y102E and D97R/E98V/Y102R mutants

To prepare D97R/E98V/Y102E mutant and D97R/E98V/Y102R mutant, PCRreaction was carried out using pcDdA-chKR127HC-RV containing D97R/E98Vas a template, and primer sets P17/P18 and P19/P20, respectively.

Vctor containing mutants prepared above are shown in Table 4.

TABLE 4 primer mutation primer position postion mutant vector HCDR3 RP17 312-279 307-309 TYr(TAC)- pcDdA-chKR12711C-RVAE F P18 295-326Glu(GAG) R P19 312-279 307-309 TYr(TAC)- pcDdA-chKR127HC-RVAR F P20295-326 Arg(CGT)

Then, D97R/E98V/Y102E mutant (hereinafter “RVAE”) and D97R/E98V/Y102Rmutant (hereinafter “RVAR”) thus obtained were measured for respectiveaffinities to antigen in according to the method described in TestExample 1.

As shown in FIG. 5, the affinity to antigen of RVAE was similar to thatof RVAA, while the affinity to antigen of RVAR was higher than that ofRVAA.

Test Example 2 Measurement of Affinity to Antigen of RVAR

The RVAR mutant prepared in step 4 of Example 4 was subjected tocompetitive ELISA to measure its affinity to antigen as follows:

COS7 cells were transfected with the plasmid prepared in step 4 ofExample 4 and the plasmid expressing chimeric light chain(pKC-dhfr-chKR127) prepared in Example 2 to produce an antibody. 5 ng ofthe antibody thus obtained was reacted with pre-S1 antigen (1×10⁻⁷ to1×10-12 M) at 37° C. for 2 hours. The resulting solution was added toeach well of a 96-well microplate coated with pre-S1 antigen and reactedat 37° C. for 30 minutes, and then the resulting solution was subjectedto ELISA analysis according to the method described in Example 4. Usedas a control is chimeric antibody (chICR127) obtained from COST cellstransfected with pcDdA-chKR127HC and pKC-dhfr-chKR127.

The affinity to antigen of RVAR was about 1.8×10⁻¹⁰ M, which is 45 timeshigher than that of chKR127, about 8.2×10-9M

Example 5 Mutation of CDR of Chimeric KR127 Antibody Light Chain byAlanine Injection

To examine the affinity of each amino acid residue of KR127 light chainLCDR1 (aa 24-34), LCDR2(aa 50-60) and LCDR3 (aa 89-97) to antigen, PCRreaction was carried out using vector pKC-dhfr-chKR127 as a template toprepare a modified gene having each amino acid residue of CDR replacedwith alanine (the replaced amino acid residue Number was indicated asKabat number) (see FIG. 2).

Forward primer YM004 of SEQ ID NO: 21 was designed to provide thesequence corresponding to the reader sequence at the 5′-end of thechimeric light chain gene and the HindIII restrition site, and a reverseprimer YM009 of SEQ ID NO: 22 was designed to have the sequencecorresponding to the N-terminal region of human light chain gene and theBsiWI(CGTACG) restriction site. These primers were used in preparationof mutants of light chain CDR residue.

YM004: 5′-CCA AAG CTT GGA AAG ATG GAT TCA CAG-3′ YM009:5′-GCA GCC ACC GTA CGT TTG ATT TCC ACC TTG GT-3′

Forward primer YM135 was designed to replace Ser26 of LCDR1 with alanine(S26A) and a reverse primer YM136, to replace AGT coding for Ser at thenucleotide Nos. 76 to 78 of LCDRI gene with GCT coding for alanine.

PCR reactions were carried out according to the method described inExample 1 except that primer sets, YM004/YM135, and YM136/YM009, wereused and that primers YM004 and YM009 were used to recombine theannealed DNA fragments obtained by PCR.

The variable region gene of the mutant thus prepared was cleaved withHind111 and BsiWI and inserted at the HinaTillBsiWI section of vectorpKC-dhfr-chKR127, to obtain pKC-dhfr-chKR12713S-S26A. The base sequenceof the modified chimeric light chain variable region gene was 5confirmed by DNA sequence analysis. The vectors containing mutantsprepared above are shown in Table 5.

In Table 5, the primer and mutation positions are numbered based on thebase sequence of SEQ ID NO: 3.

TABLE 5 primer mutation Primer position position mutant vector LCDR1 FYM135  67-102 76-78 Ser(AGT)- pKC-dhfr-chKR127BS- R YM136 86-54 Ala(GCT)S26A F YM137  69-107 79-81 Gln(CAG)- pKC-dhfr-chKR127BS- R YM138 91-56Ala(GCG Q27A F YM139  70-111 82-84 Ser(AGC)- pKC-dhfr-chKR127BS- R YM14094-58 Ala(GCC) S27aA F YM141  73-114 85-87 Leu(CTC)- pKC-dhfr-chKR127BS-R YM142 98-64 Ala(GCC) L27bA F YM143  73-116 Leu(TTA)-pKC-dhfr-chKR127BS- R YM144 102-68  88-91 Ala(GCA) L276A F YM145  79-11891-93 Tyr(TAT)- pKC-dhfr-chKR127BS- R YM146 103-69  Ala(GCA) Y27dA FYM147  83-119 94-96 Ser(AGT)- pKC-dhfr-chKR127BS- R YM148 107-69 Ala(GCT) S27eA F YM149  84-120 97-99 Asn(AAT)- pice-dhfr-chKR127BS- RYM150 110-70  Ala(GCT) N28A F YM151  88-127 100-102 Gly(GGA)-pKC-dhfr-chKR127BS- R YM152 114-74  Ala(GCA) G29A F YM153  91-130103-105 Lys(AAA)- pKC-dhfr-chKR127BS- R YM154 116-77  Ala(GCA) K30A FYM155  93-132 106-108 Thr(ACC)- pKC-dhfr-chKR127BS- R YM156 118-80 Ala(GCC) T31A F YM103  99-133 109-111 Tyr(TAT)- pKC-dhfr-chKR127BS- RYM104 120-83  Ala(GCT) Y32A F N34A-F 106-132 115-118 Asn(AAT)-pKC-dhfr-chKR127BS- R N34A-R 126-100 Ala(GCT) Y34A LCDR2 F YM129 151-188163-165 Leu(CTG)- pKC-dhfr-chKR127BS- R YM130 175-140 Ala(GCG) L50A FYM131 153-191 166-168 Val(GTG)- pKC-dhfr-chKR127BS- R YM132 179-145Ala(GCG) V51A F YM133 157-192 169-171 Ser(TCT)- pKC-dhfr-chKR127BS- RYM134 181-147 Ala(GCT) S52A F K53A-F 163-187 172-174 Lys(AAA)-pKC-dhfr-chKR127BS- R K53A-R 178-154 Ala(GCA) K53A F L54A-F 163-189175-177 Leu(CTG)- pKC-dhfr-chKR127BS- R L54A-R 180-159 Ala(GCG) LS4A FD55A-F 170-195 178-180 Asp(GAC)- pKC-dhfr-chKR127BS- R D55A-R 184-163Ala(GCC) D55A F K56A-F 175-198 181-183 Ser(TCT)- pKC-dhfr-chKR127BS- RK56A-R 190-168 Ala(GCT) S56A LCDR3 F YM113 270-304 280-282 Val(GTG)-pKC-dhfr-chKR127BS- R YM114 292-258 Ala(GCG) V89A F YM115 274-307283-285 Gln(CAA)- pKC-dhfr-chKR127BS- R YM116 294-259 Ala(GCA) Q90A FYM117 277-310 286-288 Gly(GGT)- pKC-dhfr-chKR127BS- R YM118 296-265Ala(GCT) G91A F YM119 281-310 289-291 Thr(ACA)- pKC-dhfr-chKR127BS- RYMI20 302-266 Ala(CCA) T92A F YM121 282-313 292-294 His(CAT)-pKC-dhfr-chKR127BS- R YM122 304-271 Ala(GCT) H93A F YM111 286-314295-297 Phe(TTT)- pKC-dhfr-chKR127BS- R YM112 307-274 Ala(GCT) F94A FYM123 286-317 298-300 Pro(CCT)- pKC-dhfr-chKR127BS- R YM124 308-278Ala(GCT) P95A F 114125 292-319 301-303 Gln(CAG)- pKC-dhfr-chKR127BS- RYM126 311-279 Ala(GCG) Q96A F YM127 294-320 304-306 Thr(ACG)-pKC-dhfr-chKR127BS- R YM128 313-282 Ala(GCG) T97A

Test Example 3 Measurement of Affinity to Antigen of Light Chain Mutant

COST cell was transfected with each of the light chain mutants preparedin Example 5 and the plasmid expressing chimeric heavy chain(pcDdA-chKR127HC) to produce an antibody. The antibody obtained wasmeasured for its affinity to antigen in accordance with the methoddescribed in Test Example 1.

Table 6 shows the results obtained for the mutants and pdDA-chKR127HCcontaining wildtype chimeric KR127 heavy chain.

TABLE 6 CDR mutant Kloow) L1 S26A  6.49 ± 0.244 027A 14.2 + 2.29 S27aA37.9 * 6.66 L27bA >10000 L27cA  36.8 * 11.01 Y27dA 1032.7 + 56.1 S27eA >10000 N28A >10000 G29A 23.94 * 2.62  K30A >10000 T31A 13.19 *1.98  Y32A >10000 N34A >10000 L2 LSOA 159.4 + 21.37 V51A 37.00 + 10.33S52A 14.08 * 0.509 K53A 7.928 * 0.976 L54A 12.57 + 2.453 D55A 225.2 *2.970 S56A 12.95 ± 0.367 L3 V89A 121.2 + 4.62  490A >10000 G91A >10000T92A 74.2 + 2.90 H93A 54.5 + 4.48 F94A >10000 P95A >10000 O96A 293.6 *7.13  T97A 17.3 ± 2.56

As shown in Table 6, the affinities to antigen of the mutants obtainedby replacing the Leu27b, Tyr27d, Ser27e, Asn28, Lys30, Tyr32, and Asn34of LCDR1; Leu50 and Asp55 of LCDR2; and Va189, Gln90, Gly91, Thr92, His93, Phe94, Pro95, and Gln96 of LCDR3 with alanine, respectively, weremore than 3 times lower than that of the wild type. Therefore, theseresidues was determined as SDR.

Example 6 Preparation of humanized heavy chain by SDR-grafting method

A humanized heavy chain was prepared using DP7-JH4, a human heavy chainconstructed by combining human immunoglobulin germline VH gene segmentDP7 (Tomlinson et al., J. Mol. Biol., 227, 776-798, 1992) having anamino acid sequence similar to KR127 heavy chain variable regions andhuman immunoglobulin germline JH4 segment (Ravetch et al., Cell, 27,583-591 (1981)).

The Trp33 and Asn35 in HCDR1 of the KR127 were grafted into the DP7-JH4.The Met34 in HCDR1 of the KR127 is identical to that of DP7-JIM.Further, to inhibit lowering the affinity to antigen, Tyr32 in HCDR1 ofthe KR127 was replaced with alanine of HCDR1 of a human antibody (GenBank data base 75023 (SAVVMN)).

The Arg50 and Tyr52 in HCDR2 of the KR127 were grafted onto the DP7-1H4.The Pro52a in HCDR2 of the KR127 is identical to that of DP715 JH4.

The Asp95, Tyr96, Arg97, Va198, and ArgI 02 of HCDR3 were grafted intoDP7-JH4.

Further, Ala71 and Lys73 of FR 3 (framwork region 3) in the heavy chainvariable region of KR127 antibody which affects the conformation of 20CDR loops were grafted thereto.

Then, PCR reaction was carried out using primers Ryu 166 of SEQ ID NO:23 and Hur37 of SEQ ID NO: 24 according to the method described inExample 3 to obtain a humanized heavy chain variable region gene,HuKR127VH-VII.

Ryu 166: 5′-GGA ITT GTC TGC AGT CAT TGT-GGC TCT GCC CTG GAA CTT-3′Hur 37: 5′-GAC AAA TCC ACG AGC ACA GTC TAC ATG-3′

The base sequence of the humanized heavy chain variable region gene wasdetermined by DNA sequence analysis (FIG. 2). Then, the gene was cleavedwith EcoRI and .Apal and inserted at the EcoRllApal section of vectorpdDdA-chKR127HC to obtain pHuKR127HC.

A humanzied antibody was prepared by combining humanized heavy chainthus obtained and the humanized antibody HZKR127I light chain describedin Korean. Patent No. 246128 and measured the affinity to antigen wasnumbered according to the method described in Test Example 2. Humanizedantibody HZKR127I was used as a control.

The affinity to antigen of the humanized antibody of about 1.5×10-10 Mwas about 50 times higher than that of HZKR127I, about 8.2×le M.

Example 7 Preparation of Humanized Light Chain by SDR-Grafting Method

A humanized light chain was prepared using DP7-3134, a human light chianconstructed by combining human immunoglobulin germline VK gene segmentDPK12 (Cox et al., Eur. J. Immunol., 24, 827-836 (1994)) having an aminoacid sequence similar to KR127 light chain variable regions and humanimmunoglobulin germline 0.1K4 segment (Hieter et al., J. Biol. Chem.,257, 1516-1522 (1982)).

The Tyr27d, Asn28 and Asn34 in LCDR1 of KR127 were grafted into theDPK12-3K4. The amino acid residues at position 27b, 27e, 30 and 15 32 ofDP7 is identical to those of KR127 light chain.

The Leu50 and Asp55 in LCDR2 of KR.127 were grafted into the DPK12-3K4gene.

The Va189, Gly91, Thr92, His93, Phe94, and Gln96 in LCDR3 of KR127 weregrafted into the DPK12-JK4. The residues at positions 90 and 20 95 ofDP7 is identical to those of KR127.

Further, Leu36 and Arg46 of FR 2 in the light chain variable region ofKR127 antibody (which acts on interaction with heavy chain or CDR) weregrafted thereto.

Then, PCR reaction was carried out using primers Ryul18 of SEQ ID NO: 25and Ryu 119 of SEQ ID NO: 26 according to the method described inExample 3 to prepare a humanized light chain variable region gene,HuKR127VH-IV.

Ryu 118: 5′-CTG TGG AGG CTG GCC TGG CTT CTG TAA TAA CCA-3′ Ryu 119:5′-GGC CAG CCT CCA CAG CTC CTA ATC TAT CTG-3′

The base sequence of the humanized light chain variable region gene wasdetermined by DNA sequence analysis (see HZIV of FIG. 4). Then, the genewas cleaved with HindJII and BsiWI and inserted at the HindlII/BsiWIsection of vector pKC-dhfr-chKR127BS to obtain pHuK.R.127KC.

A humanzied antibody was prepared by combining humanized light chainthus obtained and the humanized antibody HZICR1271 heavy chainde(scribed in Korean Patent No. 246128 and its affinity to antigen wasmeasured according to the method described in Test Example 2. Humanizedantibody HZKR127I was used as a control.

The affinities to antigen of the humanized antibody of about 8.4×10″⁹ Mwas similar to that of HZICR1271, about 8.2×10″⁹ M.

Example 8 Preparation of Humanized Antibody and Measurement of theAffinity to Antigen

To prepare a plasmid containing humanized heavy chain plasmid pHuKR127HCand humanized light chain plasmid pHuKR127KC, the EcoRIMpal fragmentcontaining humanized heavy chain variable region gene of pHuKR127HC andthe HindMIBsiWI fragment containing humanized light chain variableregion gene of pHuKR127KC were inserted at the EcoRIMpal and HindllBsiWIsections of vector pdCMV-dhfrC-HAVE (KCTC 10028BP), respectively, toobtain plasmid pdCIVW-dhfrC-HuKR127 (FIG. 6). E. coli DH5 a wastransformed with the plasmid thus obtained and the transformed E. coliDH5a/pdCMC-dhfrC-HuKR127 was deposited on Mar. 13, 2002 with the KoreanCollection for Type Cultures(KCTC) (Address: Korea Research Institute ofBioscience and Biotechnology(KRIBB), #52, Oun-dong, Yusong-ku, Taejon,305-333, Republic of Korea) under the accession number, KCTC 10198BP, inaccordance with the terms of Budapest Treaty on the InternationalRecognition of the Deposit of Microorganism for the Purpose of PatentProcedure.

To prepare cell line expressing the humanized antibody, dhfr-defectedCHO (chinese hamster ovary) cells were transformed with plasmidpdCMV-dhfrC-HuICR127 as follows:

CHO cells (ATCC CRL 9096) were seeded to DMEMJF12 media (GIBCO)containing 10% fetal bovine serum and subcultured in an incubator at 37°C. under an atmosphere of 5% CO₂. 5×10⁵ cells thus obtained were seededto the same media and incubated at 37° C. overnight, followed by washing3 times with OPTI-MEMI solution (GIBCO).

Meanwhile, 5 gg of the plasmid pdCMV-dhfi-C-HuICR127 was diluted in 500α of OPTI-MEIVII solution. 25 ihe of Lipofectamine was diluted in 500,u-e of the same solution. The resulting solutions were added to a 15 metube, mixed, and then, kept at room temperature for more than 15minutes. Then, 2 nit of OPTI-MEM I was added to by DNA-Lipofectaminemixture and the resulting solution was distributed evenly on the COSTcells to be kept in a 5% CO₂ incubator at 37° C. for 6 hours. Addedthereto was 3 in.c, of DMEM/F12 containing 20% fetal bovine serum andcultured for 48 hours.

Then, CHO cells were taken up with trypsin and cultured in-a-MEM media(GIBCO) of 10% dialyzed fetal bovine serum containing G418 (GIBCO BRL;550. mit) for 2 weeks. After confirming of antibody-producing ability ofthe transformed clone, the clone was cultured in a-MEM media of 10%dialyzed fetal bovine serum containing 20 nM MTX to induce amplificationof gene.

Cell line CHO/HuKR127 having the highest antibody-productivity wasselected from the clones and deposited on Mar. 13, 2002 with the KoreanCollection for Type Cultures(KCTC) under the accession number, KCTC10199BP, in accordance with the terms of Budapest Treaty on theInternational Recognition of the Deposit of Microorganism for thePurpose of Patent Procedure.

To measure the affinity to antigen of the humanized antibody HuKR127,CHO cell line thus obtained was mass cultured in a serum-absence media(CHO-SFMII, GIBCO) and subjected to protein G-shepharose 413 column(Pharmacia). Then, the antibody absorbed on the column was eluted with0.1 M glycine solution (pH 2.7) and neutralized with 1.0 M tris solution(pH 9.0), followed by dialyzing in PBS buffer (pH 7.0). Further, theaffinity to antigen of the purified antibody was determined by thecompetitive ELISA method described in Test Example 2 and compared withthat of a control, humanized HuKR1271. The result was shown in FIG. 7.

As shown in FIG. 7, the affinity to antigen of the humanized antibody ofthe present invention of 1.6×10^(I) ^(o) M was about 50 times higherthan 8.2×10⁻⁹ M3 of the control group.

Example 9 Confirmation of Immune-Response Induction of HumanizedAntibody

To confirm whether the humanized antibody of the present invention(HuKR127) prevents HAMA response, an analysis was conducted according tothe TEPITOPE method (Sturniolo et al., Nature Biotechnology, 17,555-561, 1999) to examine whether a peptide sequences which can bind toMHC (major histocompatibility complex) class II exists in the heavy andlight chain variable regions of the humanized antibody.

Tables 8a and 8b show the results of such analysis for MHC class II-5binding peptide sequences in the heavy chain variable regions of HuKR127and the light chain variable regions of HuKR127, respectively.

TABLE 7  HaKR1271 HuICR127 antiboby peptide MHC class II peptideMHC class II MHC class  LVQSGAEVV DRB1_0306 LVQSGAEVK  0 II-bindingDRB1_0307 DRB1_0308 DRB1_0311 DRB1_0421 DRB1_0701 DRB1_0703 VKPGASVKVDRB1_0102 lUtPGASVKV  0 FSSSWMNWV DRB1_0703 FTSAWMNWV  0 IfIGRIYPGDDRB1_0801 WMGRIYPSG  0 DRB1_0817 FQGRATLTA DRB1_0401 FQGRVIMTA ORB1_0305DRB1_0402 DRB1 0408 DRB1_0405 DRB1_0401 DRB1_0421 DRI31_0402 DRB1_0426DRB1_0408 DRB1_0801 DRB1_0426 DRI31_0802 DRB1_0801 DRB1_0B04 DRB1_0802DRB1_0806 DRB1_0804 DRB1_0804 DRB1_0806 DR81_0813 DRB1_0813 DRB1_0806DRB1_0617 DRBL0817 DRB1_1101 DRB1_1101 DRB1_1114 DRB1_1102 DRB1_1120DRB1 1104 DRB1_1128 DRB1-1106 DRB1_1302 DRB1_1114 DRB1_1305 DRB1_1120DRB1_1307 DRB1_1121 D/W1_1321 DRB1_1128 DRB1_1323 DRB1_1302 DRB1_1502DRB1_1305 DRB1_1307 DRB1_1311 DRB1_1321 DRB1_1322 DRB1_1323 YWGQGILVTDRB1_0401 RWGQGTLVT  0 DRB1_0405 DRB1_0421 DRB1_0426 IGRIYPGDG DRI350101MGRIYPSGG DRB1_0404 DRB5_0105 DRB1_0405 DRB1_0410 DRB1_0423 YAQKPQGRADR1_0802 YAQKFQGRV  0 VYFCAREYD DRB1_1304 VYYCAREYR DRB1_0301 YWGQGTLVTDRB1_0401 RWGQGILVT  0 DRB1_0405 DRBU1421 DRB1_0426 total 50 26

TABLE 8a  HzKR.127I HuKR127 antiboby Peptide MHC class II peptideMHC class II MHC class II- ILMTQTPLS DRBL0301 IVMTQTPLS 0 bindingDRB1_0305 DRB1_0306 DRB1 0307 DRB1_0308 DRB1_0309 DRBL0311 DRBL0401DRB1 0402 DRB1_0404 DRB1_0405 DRB1_0408 DRB1_0410 DRBL0421 DRB1 0423DREli0426 DRB1_0804 ERBL1101 DRBL1102 DRB1_1104 DRBL1106 DRB1_1107DRBL1114 DRB1_1121 DRB1_1128 DRB1_1301 DRB1_1304 DRB1_1305 DRB1_1307DRB1_1311 DREIL1321 DRB1_1322  DRB1_1323  DRB1_1327 DRB1_1328 LMTQTPLSLDRBL0101 MCIQMISL 0 DRB1_0102 DRB1_0304 MUMS ORB1_0101 wILQXPGQP 0DRB1_0305 DRBL0309 DRBL0401 DRB10408 ORM_0421 DRB10426 DRB1_0802DRB1_1101 DRB1_1107 DR/11_1114 DRB1_1120 DRB1_1128 DRB1 1302 DRB1_1305DRB1_1307 DR/a . . . 1321 DRB1_1323 DRB5_0101 DRB10105 YYCVQGTHFDRB1_0101 YYCVQGTHF DRBLOI01 DRB1_0701 DRBI_0701 DRB1_0703 DRBL0703DRB1_0104 DRB5_0101 DRB1_0105 DRB5_0105 YCVQGTHFP DR&040/ YCVQGTHFPORBL0401 DRB1_0421 DR/31_0421 ORB1_0426 DRBL0426

TABLE 8b HzKR127I HuKR127 antiboby Peptide MEC class II peptideMEC class II VGVYYCVQG DRB1 0806 VGVYYCVQG DRB1_0806 IYLVSKLDS DRB1 0301DRB1_0402 D1631-0305 DR13_0404 DRB1-0306 DRB1_0405 DRB1-0307 DRB1-0308DRB1 01301 DRB1-0309 DR131-0408 DRI31-0311 DRB1-0405 DRI31-0410ORM 01302 DR131 0806 DRB -1)423 DB1-0813 DRBI-W04 DR131-0817 DB1-110219131-1101 DRB1-11(14 DRB1-1102 DRB -1106 DRB1-1104 DRB111114 DRB1-1106IYLVSNRDS DRB -1121 DR13E1107 DRB1 1301 DR81 1114 DRB1-1307 121A3I-1120DRB1-1311 DRB -1322 DR/31-r1 a B IliP DRB1-1301 DRB1-1302 X1 T3228DRB1-1301- DR135 0101 DB1-1305 DRB5 0105 DRB1-1307 DRBE1311 DRB1 1321DRB1-1322 DRB1-1323 DRB1-1327 DRB1-1328 DRB1-1501 DR13111506 DRB1 0401DRB1-0404 DRB1-0405 DRB1 13046 _ D R B 1 0 DRDIum---nain LlYINSFID 8 0 10 DRB1 _ 1321 - DRB1 0421 8 D-0123 LIYLVSNRD DR131RB1-0126 DR1311304YLVSNRDSG 0 YLVSNRDSG DRB1 0309 40 106 total

As can be seen from Tables 7 and 8, the number of the peptide Sequencein the humanized antibody HuKR127 which binds to MHC class II was fewerthan of that the HzKR127I. These results suggest that 35 humanizedantibody HuKR127 of the present invention is expected to reduce HAMAresponse to a greater extent than HzKR127I.

While the embodiments of the subject invention have been described andillustrated, it is obvious that various changes and modifications can bemade therein without departing from the spirit of the present inventionwhich should be limited only by the scope of the appended claims.

1. (canceled)
 2. A process for preparing a humanized antibody consistingof the steps of: (a) first performing alanine scanning mutagenesis forreplacing each amino acid residue in the entire complementarilydetermining region (CDR) of a murine monoclonal antibody heavy chain andlight chain variable regions with alanine to produce a series oftransformants, selecting a transformant that has a lower affinity to thehuman antigen (KD) than of the original murine antibody, and determiningthe replaced amino acid residue of said selected transformant as aspecificity determining residue (SDR); and (b) subsequently grafting allof the said SDR to the corresponding amino acid residues into humanantibody variable regions.
 3. The process of claim 2, wherein the CDR isselected from the group consisting of HCDR1(aa 31-35), HCDR2(aa 50-65)and HCDR3(aa 95-102) of the heavy chain (SEQ ID NO: 2); and LCDR1(aa24-34), LCDR2(aa 50-56) and LCDR3(aa 89-97) of the light chain (SEQ IDNO: 4) of the murine monoclonal antibody variable regions of that bindhepatitis B virus pre-S 1 antigen, selecting a transformant that has anaffinity to antigen which is more than 3 times lower than the originalmurine antibody when replaced with alanine, determining the replacedamino acid residue of said transformant as an SDR, and grafting said SDRto the corresponding amino acid sequence in human antibody heavy chainand light chain. 4-24. (canceled)